ABSTRACT
The
introduction of new features or functions that are not present in an
original protein is a significant challenge in protein engineering. For
example, modifications to vesicular stomatitis virus glycoprotein
(VSV-G), which is commonly used to pseudotype retroviral and lentiviral
vectors for gene delivery, have been hindered by a lack of structural
knowledge of the protein. We have developed a transposon-based approach
that randomly incorporates designed polypeptides throughout a protein
to generate saturated insertion libraries and a subsequent
high-throughput selection process in mammalian cells that enables the
identification of optimal insertion sites for a novel designed
functionality. This method was applied to VSV-G in order to construct a
comprehensive library of mutants whose combined members have a
His6 tag inserted at likely every site in the original
protein sequence. Selecting the library via iterative retroviral
infections of mammalian cells led to the identification of several
VSV-G-His6 variants that were able to package high-titer
viral vectors and could be purified by Ni-nitrilotriacetic acid
affinity chromatography. Column purification of vectors reduced protein
and DNA impurities more than 5,000-fold and 14,000-fold, respectively,
from the viral supernatant. This substantially improved purity elicited
a weaker immune response in the brain, without altering the infectivity
or tropism from wild-type VSV-G-pseudotyped vectors. This work applies
a powerful new tool for protein engineering to construct novel viral
envelope variants that can greatly improve the safety and use of
retroviral and lentiviral vectors for clinical gene therapy.
Furthermore, this approach of library generation and selection can
readily be extended to other challenges in protein
engineering.
Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane.
